Methods of analyzing nucleic acids

ABSTRACT

Presented herein are methods and compositions for analyzing rare nucleic acid species. Some methods presented herein use DNA reassociation kinetics following thermal denaturation to define populations of nucleic acid sequences, e.g., highly abundant (e.g., cDNA from rRNA), moderately abundant, and less abundant or rare sequences (e.g., cDNA from mRNA).

This application is the § 371 U.S. National Stage of InternationalApplication No. PCT/US2016/034809, filed 27 May 2016, which claims thebenefit of U.S. Provisional Application No. 62/168,281, filed 29 May2015, the disclosures of which are incorporated by reference herein intheir entireties.

1 BACKGROUND

Current high throughput library preparation methods to analyze genomesand transcriptomes contain sequence information that mostly preservesthe natural abundance of genes and transcripts. Typically, genomiclibraries from higher eukaryotes, especially plants, contain asignificant amount of repetitive DNA. Similarly, transcriptome librariesfrom both eukaryotes and prokaryotes largely contain cDNA sequences thatare derived from a small number of abundant transcripts. Sequenceinformation derived from ribosomal RNA (rRNA) predominate transcriptomelibraries even after their removal prior to conversion to cDNA. Further,preparations of nucleic acids from eukaryotes, unless they arefractionated, contain sequences from organelles such as mitochondriaand/or chloroplasts and these sequences further constitute a source ofunwanted data. While useful for copy number analysis and expressionprofiling, the complexity of sequence information in these libraries maybe a hindrance in the analysis of sequence variants and may maskinformation derived from low copy genes and transcripts. Therefore,there is a need for methods to reduce the complexity of libraries toaid, for example, in the discovery and analysis of low copy genes, theirvariants and expressed transcripts.

2 BRIEF SUMMARY

In one aspect, the methods of the invention use DNA reassociationkinetics following thermal denaturation to define populations of nucleicacid sequences, e.g., highly abundant (e.g., cDNA from rRNA), moderatelyabundant, and less abundant or rare sequences (e.g., cDNA from mRNA).The rate at which a particular sequence will reassociate is proportionalto the number of copies of that sequence in the DNA sample. For example,highly-repetitive (abundant) sequence will reassociate rapidly, whilecomplex sequences (less abundant) will reassociate more slowly andremain single-stranded for a longer period of time.

In one embodiment, the methods of the invention provide for selectiveidentification and sequence analysis of both the abundant and lessabundant (e.g., rare) species of nucleic acids present in a sample. Insome embodiments, a biotinylated transposome is used to selectively tagand capture the more abundant DNA molecules in a DNA library therebyreducing the complexity of abundant sequences in the DNA library. Theless abundant DNA molecules remain in the supernatant and may be readilyprocessed for sequence analysis. The tagged and captured abundant DNAmolecules may also be readily processed for subsequent analysis.

In another embodiment, the disclosed methods use transposome-basednormalization to reduce the impact of abundant sequences (e.g., rRNA) inlibrary sequence analysis.

In one aspect, certain nucleic acids can be selectively captured from amixture of other nucleic acids and other cellular components. In someembodiments, the nucleic acids that are selectively captured arepurified from a mixture of nucleic acids and other cellular components.In some embodiments, certain nucleic acids are selectively purified froma mixture of nucleic acids and other cellular components by removing oneor more type of other nucleic acids. In some embodiments, more than onetype of nucleic acid can be selectively captured and/or purified from amixture of nucleic acids or from a biological sample.

In one aspect, the application discloses kits comprising nucleic acidbinding proteins immobilized on a solid support to selectively captureand/or purify specific types of nucleic acids from a mixture of varioustypes of nucleic acids. In some embodiments, the kits can be used topurify specific types of nucleic acids bound to the immobilized nucleicacid binding protein. In some embodiments, the kits can be used toenrich unbound nucleic acid in a mixture of nucleic acids.

In some embodiments presented herein is provided a method of analyzingrare nucleic acid species comprising: (a) providing library ofdouble-stranded nucleic acids; (b) denaturing the library; (c)renaturing the library under conditions sufficient to renature abundantnucleic acid species, wherein a portion of the library comprising lessabundant nucleic acid species does not renature; (d) contacting thelibrary with a nucleic acid binding protein that preferentially binds todouble-stranded nucleic acids; and (e) separating renatured abundantnucleic acid species from the less abundant nucleic acid species.

In certain aspects, separating comprises immobilizing binding protein toa solid support. In certain aspects, said binding protein comprises atransposase. In certain aspects, said binding protein comprises Tn5 orTS-Tn5 transposase. In certain aspects, said binding protein comprisesadaptor sequences which comprise a binding moiety capable of binding toa solid support. In certain aspects, said binding protein comprisesadaptor sequences which comprise a binding moiety capable of binding toa solid support. In certain aspects, said binding moiety comprisesbiotin and solid support comprises streptavidin. In certain aspects,binding protein comprises adaptor sequences which are not compatiblewith downstream amplification and sequencing.

In certain aspects, denaturing comprises heat denaturing. In certainaspects, heat denaturing comprises application of heat above 70° C., 75°C., 80° C., 85° C., 90° C., 91° C., 92° C., 93° C., 94° C., or above 95°C. In certain aspects, renaturing comprises lowering the denaturationtemperature to a temperature of about 40° C. to about 65° C. for asufficient period of time to renature abundant nucleic acid species. Incertain aspects, said sufficient period of time comprises about 30minutes to about 24 hours. In certain aspects, denaturing compriseschemical denaturing.

In certain aspects, the method further comprises sequencing theseparated less abundant nucleic acid species. In certain aspects, thelibrary comprises genomic DNA. In certain aspects, the library comprisesrandomly sheared DNA. In certain aspects, the library comprisesdouble-stranded cDNA. In certain aspects, the nucleic acids in thelibrary comprise adaptor sequences ligated to the ends of the nucleicacids. In certain aspects, the adaptor sequences comprise one or moreof: amplification priming binding region, sequencing primer bindingregion, and promotor sequences for in vitro transcription. In certainaspects, said abundant nucleic acid species comprise highly repetitivesequences. In certain aspects, said library comprises nucleic acid froma single cell or a small group of cells. In certain aspects, saidabundant nucleic acid species comprise host sequences and said lessabundant nucleic acid species comprise pathogen content. In certainaspects, said abundant nucleic acid species comprise duplicate librariesin a sequencing library. In certain aspects, the library comprisesgenomic RNA.

3 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a schematic diagram of an example of a forktailtransposon;

FIG. 1B illustrates a schematic diagram of assembling a Ts-Tn5 suicidetransposome complex;

FIG. 2A shows a plot of the fragment size distribution of tagmented DNAprepared using transposomes comprising a standard TotalScript transposonand a standard Tn5 transposase;

FIG. 2B shows a plot of the fragment size distribution of tagmented DNAprepared using transposomes comprising the forktail transposon of FIG. 1and a standard Tn5 transposase;

FIG. 2C shows a plot of the fragment size distribution of tagmented DNAprepared using transposomes comprising the standard TotalScripttransposon and Ts-Tn5 transposase;

FIG. 2D shows a plot of the fragment size distribution of tagmented DNAprepared using transposomes comprising the forktail transposon of FIG. 1and Ts-Tn5 transposase;

FIG. 3A shows a plot of the fragment size distribution of tagmented DNAprepared at elevated (>55° C.) temperatures using Ts-Tn5 transposomes;

FIG. 3B shows a plot of the fragment size distribution of tagmented DNAprepared at elevated (>55° C.) temperatures using a standard Tn5transposome (TDE1);

FIG. 4 shows a plot of DNA fragment size verses tagmentation reactiontemperature for the tagmented DNA of FIG. 3A and FIG. 3B;

FIG. 5 illustrates a flow diagram of an example of a method of reducingthe abundance of double-stranded DNA in a complex library bysimultaneously fragmenting and tagging the end(s) of double-stranded DNAusing a transposome;

FIG. 6 shows a photograph of an agarose gel of the fragmentation ofsingle-stranded M13mp19 DNA and double-stranded pUC19 DNA by EZ-Tn5™ME-Transposome;

FIG. 7 shows a photograph of an agarose gel of the specificfragmentation of double-stranded pUC19 DNA in a mixture comprisingsingle-stranded M13mp19 DNA and double-stranded pUC19 DNA;

FIG. 8 illustrates a flow diagram of an example of a method of preparingand normalizing an RNA library for sequencing;

FIG. 9 illustrates a schematic diagram showing pictorially the steps ofthe method of FIG. 8;

FIG. 10 shows a bar graph of the percent sequence alignment of controland suicide-transposome normalized libraries of Table 2, % aligned meansalignment to genome, including known transcripts, non-coding RNA andintergenic sequence;

FIG. 11 shows a bar graph of the percent rRNA, mitochondrial RNA andduplicate reads in the abundant sequence fraction of samples of FIG. 10;

FIG. 12 shows a panel of the read distributions in control andsuicide-transposome normalized libraries of Table 2; and

FIG. 13 shows a panel of the alignment locations in control andsuicide-transposome normalized libraries of Table 2.

4 DESCRIPTION

The presently disclosed subject matter is related to U.S. Patent Pub.No. 20050153333, entitled “Selective terminal tagging of nucleic acids,”published on Jul. 14, 2005; U.S. Patent Pub. No. 20100297643, entitled“Terminus-specific DNA modification using random-sequence templateoligonucleotides,” published on Nov. 25, 2010; and U.S. Patent Pub. No.20140194324, entitled “Sample preparation of a solid support,” publishedon Jul. 10, 2014, the entire disclosures of which are incorporatedherein by reference.

As used herein the term “at least a portion” and/or grammaticalequivalents thereof can refer to any fraction of a whole amount. Forexample, “at least a portion” can refer to at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% of awhole amount.

As used herein, the term “about” means+/−10%.

As used herein, the term “elevated temperature” means a temperatureabove 40° C. In some embodiments, the elevated temperature is within therange of about: 40° C.-95° C., 45° C.-90° C., or 50° C.-70° C. In someembodiments, the elevated temperature is about 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95° C., or more.

As used herein, the terms “Affinity binding substances” or “affinitybinding molecules” or “affinity molecules” mean molecules that haveaffinity for and “bind” to each other under certain conditions, referredto as “binding conditions”, to form a “specific binding pair.” Forexample, biotin and streptavidin, biotin and avidin, or digoxigenin anda specific antibody that binds digoxigenin are examples of “specificbinding pairs,” with the members of each specific binding paircomprising “affinity binding molecules” or “affinity binding substances”or “affinity molecules.” Affinity binding molecules (e.g., biotin and/orstreptavidin) can be covalently joined or conjugated, or non-covalentlybound, to other molecules (e.g., to RNA or DNA) or to a solid surfaceusing methods known in the art (e.g., using reagents and methods asdescribed in Avidin-Biotin Chemistry: A Handbook, by D. Savage et al.,Pierce Chemical Company, 1992, and in Handbook of Fluorescent Probes andResearch Products, Ninth Edition, by R. P. Hoagland, Molecular Probes,Inc., and in BIOCONJUGATE Techniques, by Greg T. Hermanson, Published byAcademic Press, Inc., San Diego, Calif., 1996). Affinity molecules thatare conjugated to DNA or RNA can also be synthesized using anoligonucleotide synthesizer using reagents and methods known in the art.The term “binding” according to the present invention means theinteraction between an affinity molecule and an affinity bindingsubstance as a result of non-covalent bonds, such as, but not limitedto, hydrogen bonds, hydrophobic interactions, van der Waals bonds, andionic bonds. Without being bound by theory, it is believed in the artthat these kinds of non-covalent bonds result in binding, in part due tocomplementary shapes or structures of the molecules involved in thespecific binding pair. Based on the definition for “binding,” and thewide variety of affinity binding molecules or specific binding pairs, itis clear that binding conditions vary for different specific bindingpairs. Those skilled in the art can easily find or determine conditionswhereby, in a sample, binding occurs between the affinity bindingmolecules. In particular, those skilled in the art can easily determineconditions whereby binding between affinity binding molecules that wouldbe considered in the art to be “specific binding” can be made to occur.As understood in the art, such specificity is usually due to the higheraffinity between the affinity binding molecules than for othersubstances and components (e.g., vessel walls, solid supports) in asample. In certain cases, the specificity might also involve, or mightbe due to, a significantly more rapid association of affinity bindingmolecules than with other substances and components in a sample.

The terms “tag” and “tag domain” as used herein refer to a portion ordomain of a polynucleotide that exhibits a sequence for a desiredintended purpose or application. Some embodiments presented hereininclude a transposome complex comprising a polynucleotide having a 3′portion comprising a transposon end sequence, and tag comprising a tagdomain. Tag domains can comprise any sequence provided for any desiredpurpose. For example, in some embodiments, a tag domain comprises one ormore restriction endonuclease recognition sites. In some embodiments, atag domain comprises one or more regions suitable for hybridization witha primer for a cluster amplification reaction. In some embodiments, atag domain comprises one or more regions suitable for hybridization witha primer for a sequencing reaction. It will be appreciated that anyother suitable feature can be incorporated into a tag domain. In someembodiments, the tag domain comprises a sequence having a length between5 and 200 bp. In some embodiments, the tag domain comprises a sequencehaving a length between 10 and 100 bp. In some embodiments, the tagdomain comprises a sequence having a length between 20 and 50 bp. Insome embodiments, the tag domain comprises a sequence having a lengthbetween 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 and200 bp.

As used herein, the term “target nucleic acid” means any nucleic acid ofinterest.

Nucleic Acid

As used herein, nucleic acids can include single stranded, doublestranded, and/or partially double stranded DNA; single stranded, doublestranded, and/or partially double stranded cDNA; products of wholegenome amplification (WGA); single stranded, double stranded; and/orpartially double stranded RNA; peptide nucleic acid, morpholino nucleicacid, locked nucleic acid, glycol nucleic acid, threose nucleic acid,mixed samples of nucleic acids, polyploidy DNA (i.e., plant DNA),mixtures thereof, and hybrids thereof. In a preferred embodiment,genomic DNA fragments or amplified copies thereof are used as nucleicacids. In another preferred embodiment, cDNA, mitochondrial DNA orchloroplast DNA is used.

Nucleic acid can comprise any nucleotide sequence. In some embodiments,the nucleic acid comprises homopolymer sequences. A nucleic acid canalso include repeat sequences. Repeat sequences can be any of a varietyof lengths including, for example, 2, 5, 10, 20, 30, 40, 50, 100, 250,500 or 1000 nucleotides or more. Repeat sequences can be repeated,either contiguously or non-contiguously, any of a variety of timesincluding, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times ormore.

In some embodiments, the length of the nucleic acids is about 20 bp, 30bp, 40 bp, 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300bp, 2400 bp, 2500 bp, 2600 bp, 2700 bp, 2800 bp, 2900 bp, 3000 bp, 3100bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700bp, 4800 bp, 4900 bp, 5000 bp, 10000 bp, 30000 bp, 40000 bp, 50000 bp,60000 bp, 70000 bp, 80000 bp, 90000 bp, 100 kbp (kilo base pair), 200kbp, 300 kbp, 400 kbp, 500 kbp, 600 kbp, 700 kbp, 800 kbp, 900 kbp, 1mbp (mega base pair), 2 mbp, 3 mbp, 4 mbp, 5 mbp, 6 mbp, 7 mbp, 8 mbp, 9mbp, 10 mbp or longer.

Some embodiments described herein can utilize a single target nucleicacid. Other embodiments can utilize a plurality of target nucleic acids.In such embodiments, a plurality of target nucleic acids can include aplurality of the same target nucleic acids, a plurality of differenttarget nucleic acids where some target nucleic acids are the same, or aplurality of target nucleic acids where all target nucleic acids aredifferent. Embodiments that utilize a plurality of target nucleic acidscan be carried out in multiplex formats so that reagents are deliveredsimultaneously to the target nucleic acids, for example, in one or morechambers or on an array surface. In some embodiments, the plurality oftarget nucleic acids can include substantially all of a particularorganism's genome. The plurality of target nucleic acids can include atleast a portion of a particular organism's genome including, forexample, at least about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%,or 99% of the genome. In particular embodiments the portion can have anupper limit that is at most about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%,90%, 95%, or 99% of the genome

Nucleic acids can be obtained from any source. For example, nucleicacids may be prepared from nucleic acid molecules obtained from a singleorganism or from populations of nucleic acid molecules obtained fromnatural sources that include one or more organisms. Sources of nucleicacid molecules include, but are not limited to, organelles, cells,tissues, organs, or organisms. Cells that may be used as sources ofnucleic acid molecules may be prokaryotic (bacterial cells, for example,Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Thermus aquaticus, Thermococcus litoralis, andStreptomyces genera); archeaon, such as crenarchaeota, nanoarchaeota oreuryarchaeotia; or eukaryotic such as fungi, (for example, yeasts),plants, protozoans and other parasites, and animals (including insects(for example, Drosophila spp.), nematodes (e.g., Caenorhabditiselegans), and mammals (for example, rat, mouse, monkey, non-humanprimate and human).

Nucleic acid may be obtained from a biological sample. The term“biological sample” as used herein includes samples such as celllysates, intact cells, organisms, organs, tissues and bodily fluids.“Bodily fluids” may include, but are not limited to, blood, dried blood,clotted blood, serum, plasma, saliva, cerebral spinal fluid, pleuralfluid, tears, lactal duct fluid, lymph, sputum, urine, amniotic fluid,and semen. A sample may include a bodily fluid that is “acellular.” An“acellular bodily fluid” includes less than about 1% (w/w) wholecellular material. Plasma or serum are examples of acellular bodilyfluids. A sample may include a specimen of natural or synthetic origin(i.e., a cellular sample made to be acellular).

In some embodiments, the biological sample can be from a human or from anon-human origin. In some embodiments, the biological sample can be froma human patient. In some embodiments, the biological sample can be froma newborn human.

The term “Plasma” as used herein refers to acellular fluid found inblood. “Plasma” may be obtained from blood by removing whole cellularmaterial from blood by methods known in the art (e.g., centrifugation,filtration, and the like).

Nucleic acids can be enriched for certain sequences of interest usingvarious methods well known in the art. Examples of such methods areprovided in Int. Pub. No. WO/2012/108864, which is incorporated hereinby reference in its entirety.

Nucleic Acid Binding Protein

As used herein, the term “nucleic acid binding protein” means a proteinbinds to nucleic acids, for example, single stranded, double stranded,and/or partially double stranded DNA; single stranded, double stranded,and/or partially double stranded cDNA; products of whole genomeamplification (WGA); single stranded, double stranded; and/or partiallydouble stranded RNA; peptide nucleic acid, morpholino nucleic acid,locked nucleic acid, glycol nucleic acid, threose nucleic acid, mixedsamples of nucleic acids, polyploidy DNA (i.e., plant DNA), mixturesthereof, and hybrids thereof.

In some embodiments, the nucleic acid binding protein binds specificallyto double stranded nucleic acids. In some embodiments, the nucleic acidbinding protein binds specifically to double stranded DNA. In someembodiments, the nucleic acid binding protein binds specifically todouble stranded RNA. In some embodiments, the nucleic acid bindingprotein binds specifically to single stranded DNA. In some embodiments,the nucleic acid binding protein binds specifically to single strandedRNA. In some embodiments, the nucleic acid binding protein binds tospecific nucleic acid sequences, for example, certain restrictionendonucleases.

In some embodiments, the nucleic acid binding protein has enzymaticactivity. In some embodiments, the enzymatic activity may be a DNAaltering activity such a nuclease activity, recombinase activity, ligaseactivity, kinase activity, gyrase activity, polymerase activity,transposase activity. In some embodiments, the nucleic acid bindingprotein requires cofactors, for example, ATP, NADP,S-adenosyl-L-methionine, metal ions, e.g., Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺,Al³⁺. In some embodiments, the enzymatic activity requires cofactors. Insome embodiments, the nucleic acid binding protein can bind to nucleicacids in the absence of cofactors. In some embodiments, the nucleic acidbinding protein with an enzymatic activity can bind to the nucleic acidin the absence of cofactors.

In some embodiments, the nucleic acid binding protein can be from athermophilic organisms, for example, Thermus aquaticus, Thermococcuslitoralis. Examples of other thermophilic organisms are disclosed in M TMadigan, J M Martinko & J Parker (1997) Brock Biology of Microorganisms.Eighth edition. Prentice Hall; Extremophiles; Special issue ofFederation of European Microbiological Societies (FEMS) MicrobiologyReviews 18, Nos. 2-3; May 1996, which are incorporated by referenceherein in its entirety. In some embodiments, the nucleic acid bindingprotein can be modified to withstand elevated temperature. In someembodiments, the nucleic acid binding proteins can be fused with otherproteins or peptides such that the altered protein are stable atelevated temperature.

Exemplary nucleic acid binding proteins that binds to double strandedDNA include, but are not limited to: transposases, restrictionendonucleases, transcription factors, DNA dependent RNA polymerases.

Exemplary restriction endonucleases are provided in the Table below.

Recognition Enzyme Source Sequence Cut EcoRI Escherichia coli 5′GAATTC5′---G     AATTC---3′ 3′CTTAAG 3′---CTTA      G---5′ EcoRIEscherichia coli 5′CCWGG 5′---   CCWGG---3′ 3′GGWCC 3′---GGWCC   ---5′BamHI Bacillus 5′GGATCC 5′---G   GATCC---3′ amyloliquefaciens 3′CCTAGG3′---CCTAG   G---5′ HindIII Haemophilus 5′AAGCTT 5′---A    AGCTT---3′influensae 3′TTCGAA 3---TTCGA  A---5′ TagI Thermus aquaticus 5′TCGA5′---T   CGA---3′ 3′AGCT 3′---AGC   T---5′ NotI Nocardia otitidis5′GCGGCCGC 5′---GC   GGCCGC---3′ 3′CGCCGGCG 3′---CGCCGG  CG---5′HinFI:″Hin″FI Haemophilus 5′GANTC 5′---G   ANTC---3′ influenzae 3′CTNAG3′---CTNA   G---5′ Sau3AI Staphylococcus 5′GATC 5′---   GATC---3′ aureus3′CTAG 3′---CTAG   ---5′ PvuII* Proteus vulgaris 5′CAGCTG5′---CAG   CTG---3′ 3′GTCGAC 3′---GTC   GAC---5′ SmaI*Serratia marcescens 5′CCCGGG 5′---CCC   GGG---3′ 3′GGGCCC3′---GGG   CCC---5′ HaeIII* Haemophilus 5′GGCC 5′---GG   CC---3′aegyptius 3′CCGG 3′---CC   GG---5′ HgaI^([68]) Haemophilus 5′GACGC5′---NN   NN---3′ gallinarum 3′CTGCG 3′---NN   NN---5′ AluI*Arthrobacter luteus 5′AGCT 5′---AG   CT---3′ 3′TCGA 3′---TC   GA---5′EcoRV* Escherichia coli 5′GATATC 5′---GAT   ATC---3′ 3′CTATAG3′---CTA   TAG---5′ EcoP15I Escherichia coli 5′CAGCAGN₂₅NN5′---CAGCAGN₂₅   NN---3′ 3′GTCGTCN₂₅NN 3′---GTCGTCN₂₅NN   ---5′KpnI^([69]) Klebsiella 5′GGTACC 5′---GGTAC   C---3′ pneumoniae 3′CCATGG3′---C   CATGG---5′ PstI^([69]) Providencia 5′CTGCAG 5′---CTGCA   G---3′stuartii 3′GACGTC 3′---G   ACGTC---5′ SacI^([69]) Streptomyces 5′GAGCTC5′---GAGCT   C---3′ achromogenes 3′CTCGAG 3′---C   TCGAG---5′SalI^([69]) Streptomyces 5′GTCGAC 5′---G   TCGAC---3′ albus 3′CAGCTG3′---CAGCT   G---5′ ScaI*^([69]) Streptomyces 5′AGTACT5′---AGT   ACT---3′ caespitosus 3′TCATGA 3′---TCA   TGA---5′ SpeISphaerotilus 5′ACTAGT 5′---A   CTAGT---3′ natans 3′TGATCA3′---TGATC   A---5′ SphI^([69]) Streptomyces 5′GCATGC5′---GCATG   C---3′ phaeochromogenes 3′CGTACG 3′---C   GTACG---5′StuO*^([70][71]) Streptomyces 5′AGGCCT 5′---AGG   CCT---3′ tubercidicua3′TCCGGA 3′---TCC   GGA---5′ XbaI^([69]) Xanthomonas 5′TCTAGA5′---T   CTAGA---3′ badrii 3′AGATCT 3′---AGATC  T---5′

The restriction endonucleases are available commercially through NewEngland Biolabs, Inc., MA, USA.

Exemplary transposases include but are not limited to transposases fromTn5 (NCBI Ref No. U00004.1, variants of Tn5), Thermus aquaticus (NCBIRef No. WP_003044334.1).

Exemplary nucleic acid binding proteins that bind to single stranded DNAinclude single stranded DNA binding proteins (SSB) of Escherichia coli(NCBI Ref No. WP_012846861.1), bacteria phage T7 (NCBI Ref No.NP_041970.1), bacteria phage T5 (NCBI Ref No. YP_006950.1); replicationprotein A of Xenopus laevis (NCBI Ref No. NP_001081585.1), Mus musculus(NCBI Ref No. NP_080929.1), Drosophila melanogaster (NCBI Ref No.NP_524274.1), Thermus aquaticus (NCBI Ref No. EED09986.1), Homo sapiens(NCBI Ref No. NP_002936.1).

Exemplary nucleic acid binding proteins that bind to both singlestranded and double stranded DNA include but are not limited toEscherichia coli recA (NCBI Ref No. YP_006777928.1), Thermus aquaticusrecA (NCBI Ref No. WP_003043690.1).

Exemplary nucleic acid binding proteins that bind to RNA include but arenot limited to RNA binding proteins, ribonucleases such as RNase A(binds to single stranded RNA), RNase III, RNase L, RNase P, RNase T1(binds to single stranded RNA), RNase T2 (binds to single stranded RNA),RNase U2 (binds to single stranded RNA).

Exemplary nucleic acid binding proteins that bind to RNA-DNA hybridinclude but are not limited to RNase H (e.g., HIV-1 RNase H, MMLV RNaseH), Reverse transcriptases.

Transposomes

A “transposome” comprises an integration enzyme such as an integrase ortransposase, and a nucleic acid comprising an integration recognitionsite, such as a transposase recognition site. In embodiments providedherein, the transposase can form a functional complex with a transposaserecognition site that is capable of catalyzing a transposition reaction.The transposase may bind to the transposase recognition site and insertthe transposase recognition site into a target nucleic acid in a processsometimes termed “tagmentation”. In some such insertion events, onestrand of the transposase recognition site may be transferred into thetarget nucleic acid. In one example, a transposome comprises a dimerictransposase comprising two subunits, and two non-contiguous transposonsequences. In another example, a transposome comprises a transposasecomprises a dimeric transposase comprising two subunits, and acontiguous transposon sequence.

Exemplary transposases include, but are not limited to Mu, Tn10, Tn5,hyperactive Tn5 (Goryshin and Reznikoff, J. Biol. Chem., 273:7367(1998)).

Some embodiments can include the use of a hyperactive Tn5 transposaseand a Tn5-type transposase recognition site (Goryshin and Reznikoff, J.Biol. Chem., 273:7367 (1998)), or MuA transposase and a Mu transposaserecognition site comprising R1 and R2 end sequences (Mizuuchi, K., Cell,35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Anexemplary transposase recognition site that forms a complex with ahyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase, EpicentreBiotechnologies, Madison, Wis.) comprises the following 19b transferredstrand (sometimes “M” or “ME”) and non-transferred strands: 5′AGATGTGTATAAGAGACAG 3′, 5′ CTGTCT CTTATACACATCT 3′, respectively. MEsequences can also be used as optimized by a skilled artisan.

More examples of transposition systems that can be used with certainembodiments of the compositions and methods provided herein includeStaphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183: 2384-8,2001; Kirby C et al., Mol. Microbiol., 43: 173-86, 2002), Ty1 (Devine &Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and InternationalPublication WO 95/23875), Transposon Tn7 (Craig, N L, Science. 271:1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol.,204:27-48, 1996), Tn/O and IS10 (Kleckner N, et al., Curr Top MicrobiolImmunol., 204:49-82, 1996), Mariner transposase (Lampe D J, et al., EMBOJ., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr. Topics Microbiol.Immunol., 204: 125-43, 1996), P Element (Gloor, G B, Methods Mol. Biol.,260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32,1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top.Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et al., ProcNatl Acad Sci USA, 86:2525-9, 1989), and retrotransposon of yeast (Boeke& Corces, Annu Rev Microbiol. 43:403-34, 1989). More examples includeIS5, Tn10, Tn903, IS911, Sleeping Beauty, SPIN, hAT, PiggyBac, Hermes,TcBuster, AeBuster1, Tol2, and engineered versions of transposase familyenzymes (Zhang et al., (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct. 16;Wilson C. et al (2007) J. Microbiol. Methods 71:332-5).

Variants of Tn5 transposases, such as having amino acid substitutions,insertions, deletions, and/or fusions with other proteins or peptidesare disclosed in U.S. Pat. Nos. 5,925,545; 5,965,443; 7,083,980;7,608,434; and U.S. patent application Ser. No. 14/686,961. The patentsand the patent application are incorporated herein by reference in itsentirety. In some embodiments, the Tn5 transposase comprise one or moresubstitutions at positions 54, 56, 372, 212, 214, 251, and 338 withrespect to the wild type protein as disclosed in U.S. patent applicationSer. No. 14/686,961. In some embodiments, the Tn5 wild-type protein orits variant can further comprise a fusion polypeptide. In someembodiments, the polypeptide domain fused to the transposase cancomprise, for example, Elongation Factor Ts. Exemplary Tn5 transposasesand its variants are shown in SEQ ID NOs: 1-22.

More examples of integrases that may be used with the methods andcompositions provided herein include retroviral integrases and integraserecognition sequences for such retroviral integrases, such as integrasesfrom HIV-1, HIV-2, SIV, PFV-1, RSV.

In one aspect, the invention provides methods of reducing the complexityof a nucleic acid library. The nucleic acid library may be, for example,a genomic DNA library or a double-stranded cDNA library prepared usingmRNA or total RNA. In various embodiments, the methods of the inventionuse a modified Tn5 transposase to remove double-stranded DNA moleculesfrom a mixture of single- and double-stranded DNA molecules to reducethe complexity of a DNA library. In one example, the transposase isTs-Tn5. In another example, the transposase is EZ-Tn5™ (Illumina, Inc.).

In various embodiments, the methods of the invention use DNAreassociation kinetics following thermal denaturation to definepopulations of nucleic acid sequences, e.g., highly abundant (e.g., cDNAfrom rRNA), moderately abundant, and less abundant or rare sequences(e.g., cDNA from mRNA). The rate at which a particular sequence willreassociate is proportional to the number of copies of that sequence inthe DNA sample. For example, highly-repetitive (abundant) sequence willreassociate rapidly, while complex sequences (less abundant) willreassociate more slowly and remain single-stranded for a longer periodof time.

In one embodiment, the methods of the invention provide for selectiveidentification and sequence analysis of both the abundant and lessabundant (e.g., rare) species of nucleic acids present in a sample. Inone example, a biotinylated transposome is used to selectively tag andcapture the more abundant DNA molecules in a DNA library therebyreducing the complexity of abundant sequences in the DNA library. Theless abundant DNA molecules remain in the supernatant and may be readilyprocessed for sequence analysis. The tagged and captured abundant DNAmolecules may also be readily processed for subsequent analysis.

In another embodiment, the methods of the invention usetransposome-based normalization to reduce the impact of abundantsequences (e.g., rRNA) in library sequence analysis.

In one application, Ts-Tn5 fusion transposases are used in thepreparation of directional RNA-seq libraries for sequencing on nextgeneration sequencing platforms (e.g., Illumina GAIIx or HiSeqplatforms).

Solid Support

In some embodiments, the solid support or its surface is non-planar,such as the inner or outer surface of a tube or vessel. In someembodiments, the solid support comprises microspheres or beads. By“microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. Suitable bead compositionsinclude, but are not limited to, plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose, cellulose, nylon, cross-linked micelles and teflon, aswell as any other materials outlined herein for solid supports may allbe used. “Microsphere Detection Guide” from Bangs Laboratories, FishersInd. is a helpful guide. In certain embodiments, the microspheres aremagnetic microspheres or beads. In some embodiments, the beads can becolor coded. For example, MicroPlex® Microspheres from Luminex, Austin,Tex. may be used. In some embodiments, the solid support compriseaffinity binding molecules. Exemplary affinity binding moleculesinclude, but are not limited to biotin-streptavidin, antigen-antibody,enzyme-substrate. In some embodiments, the beads comprise streptavidin.

The beads need not be spherical; irregular particles may be used.Alternatively or additionally, the beads may be porous. The bead sizesrange from nanometers, i.e. about 10 nm, to millimeters in diameter,i.e. 1 mm, with beads from about 0.2 micron to about 200 microns beingpreferred, and from about 0.5 to about 5 micron being particularlypreferred, although in some embodiments smaller or larger beads may beused. In some embodiments, beads can be about 0.1, 0.2, 0.3, 0.4, 0.5.0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 200 μm in diameter.

Immobilizing Nucleic Acid Proteins to Solid Support

Methods for immobilizing proteins to solid support by covalent bonds arewell known in the art. For example, a variety of surface chemistries canbe used to immobilize a binding protein to a solid surface includingcovalent bonding of amine groups on proteins to aldehyde or epoxidegroups on silanized glass surfaces, or other functional groups on asolid support (see Guo and Zhu, (2007) “The Critical Role of SurfaceChemistry in Protein Microarrays” in Functional Protein Microarrays inDrug Discovery, Ed. Paul F. Predki, CRC Press, Chapter 4, pgs 53-71).Additionally, known methods can be used to attach a protein moiety to asolid support including, for example, as described in U.S. Pat. Nos.8,022,013, 8,912,130, 7,977,476, 7,259,258, U.S. Patent ApplicationPublication No. 20130059741, and PCT application publicationWO/2001/041918, each of which is incorporated by reference in itsentirety.

In some embodiments, the proteins, such as nucleic acid binding proteinscan be immobilized on a solid support by non-covalent means, for exampleby the use of affinity binding molecules. In some embodiments, thenucleic acid binding protein is transposase. The transposons in thetransposome complex may comprise biotin and the transposome complex canbe immobilized on a solid support comprising streptavidin orneutravidin.

4.1 Sequencing Methods

The methods described herein can be used in conjunction with a varietyof nucleic acid sequencing techniques. Particularly applicabletechniques are those wherein nucleic acids are attached at fixedlocations in an array such that their relative positions do not changeand wherein the array is repeatedly imaged. Embodiments in which imagesare obtained in different color channels, for example, coinciding withdifferent labels used to distinguish one nucleotide base type fromanother are particularly applicable. In some embodiments, the process todetermine the nucleotide sequence of a target nucleic acid can be anautomated process. Preferred embodiments include sequencing-by-synthesis(“SBS”) techniques.

“Sequencing-by-synthesis (“SBS”) techniques” generally involve theenzymatic extension of a nascent nucleic acid strand through theiterative addition of nucleotides against a template strand. Intraditional methods of SBS, a single nucleotide monomer may be providedto a target nucleotide in the presence of a polymerase in each delivery.However, in the methods described herein, more than one type ofnucleotide monomer can be provided to a target nucleic acid in thepresence of a polymerase in a delivery.

SBS can utilize nucleotide monomers that have a terminator moiety orthose that lack any terminator moieties. Methods utilizing nucleotidemonomers lacking terminators include, for example, pyrosequencing andsequencing using γ-phosphate-labeled nucleotides, as set forth infurther detail below. In methods using nucleotide monomers lackingterminators, the number of nucleotides added in each cycle is generallyvariable and dependent upon the template sequence and the mode ofnucleotide delivery. For SBS techniques that utilize nucleotide monomershaving a terminator moiety, the terminator can be effectivelyirreversible under the sequencing conditions used as is the case fortraditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methodsdeveloped by Solexa (now Illumina, Inc.).

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of thenucleotide, such as release of pyrophosphate; or the like. Inembodiments, where two or more different nucleotides are present in asequencing reagent, the different nucleotides can be distinguishablefrom each other, or alternatively, the two or more different labels canbe the indistinguishable under the detection techniques being used. Forexample, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriateoptics as exemplified by the sequencing methods developed by Solexa (nowIllumina, Inc.).

Preferred embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, the disclosures of which are incorporatedherein by reference in their entireties). In pyrosequencing, releasedPPi can be detected by being immediately converted to adenosinetriphosphate (ATP) by ATP sulfurylase, and the level of ATP generated isdetected via luciferase-produced photons. The nucleic acids to besequenced can be attached to features in an array and the array can beimaged to capture the chemiluminscent signals that are produced due toincorporation of a nucleotides at the features of the array. An imagecan be obtained after the array is treated with a particular nucleotidetype (e.g., A, T, C or G). Images obtained after addition of eachnucleotide type will differ with regard to which features in the arrayare detected. These differences in the image reflect the differentsequence content of the features on the array. However, the relativelocations of each feature will remain unchanged in the images. Theimages can be stored, processed and analyzed using the methods set forthherein. For example, images obtained after treatment of the array witheach different nucleotide type can be handled in the same way asexemplified herein for images obtained from different detection channelsfor reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in International Patent Pub. No. WO 04/018497 and U.S. Pat. No.7,057,026, the disclosures of which are incorporated herein byreference. This approach is being commercialized by Solexa (now IlluminaInc.), and is also described in International Patent Pub. No. WO91/06678 and International Patent Pub. No. WO 07/123,744, each of whichis incorporated herein by reference. The availability offluorescently-labeled terminators in which both the termination can bereversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

Preferably in reversible terminator-based sequencing embodiments, thelabels do not substantially inhibit extension under SBS reactionconditions. However, the detection labels can be removable, for example,by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. Inparticular embodiments, each cycle involves simultaneous delivery offour different nucleotide types to the array and each nucleotide typehas a spectrally distinct label. Four images can then be obtained, eachusing a detection channel that is selective for one of the fourdifferent labels. Alternatively, different nucleotide types can be addedsequentially and an image of the array can be obtained between eachaddition step. In such embodiments each image will show nucleic acidfeatures that have incorporated nucleotides of a particular type.Different features will be present or absent in the different images duethe different sequence content of each feature. However, the relativeposition of the features will remain unchanged in the images. Imagesobtained from such reversible terminator-SBS methods can be stored,processed and analyzed as set forth herein. Following the image capturestep, labels can be removed and reversible terminator moieties can beremoved for subsequent cycles of nucleotide addition and detection.Removal of the labels after they have been detected in a particularcycle and prior to a subsequent cycle can provide the advantage ofreducing background signal and crosstalk between cycles. Examples ofuseful labels and removal methods are set forth below.

In particular embodiments some or all of the nucleotide monomers caninclude reversible terminators. In such embodiments, reversibleterminators/cleavable fluors can include fluor linked to the ribosemoiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005),which is incorporated herein by reference). Other approaches haveseparated the terminator chemistry from the cleavage of the fluorescencelabel (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), whichis incorporated herein by reference in its entirety). Ruparel et aldescribed the development of reversible terminators that used a small 3′allyl group to block extension, but could easily be deblocked by a shorttreatment with a palladium catalyst. The fluorophore was attached to thebase via a photocleavable linker that could easily be cleaved by a 30second exposure to long wavelength UV light. Thus, either disulfidereduction or photocleavage can be used as a cleavable linker. Anotherapproach to reversible termination is the use of natural terminationthat ensues after placement of a bulky dye on a dNTP. The presence of acharged bulky dye on the dNTP can act as an effective terminator throughsteric and/or electrostatic hindrance. The presence of one incorporationevent prevents further incorporations unless the dye is removed.Cleavage of the dye removes the fluor and effectively reverses thetermination. Examples of modified nucleotides are also described in U.S.Pat. Nos. 7,427,673, and 7,057,026, the disclosures of which areincorporated herein by reference in their entireties.

Additional exemplary SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentPub. No. 2007/0166705, U.S. Patent Pub. No. 2006/0188901, U.S. Pat. No.7,057,026, U.S. Patent Pub. No. 2006/0240439, U.S. U.S. Patent Pub. No.2006/0281109, International Patent Pub. No. WO 05/065814, U.S. PatentPub. No. 2005/0100900, International Patent Pub. No. WO 06/064199,International Patent Pub. No. WO 07/010,251, U.S. U.S. Patent Pub. No.2012/0270305 and U.S. Patent Pub. No. 2013/0260372, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize detection of four different nucleotidesusing fewer than four different labels. For example, SBS can beperformed utilizing methods and systems described in the incorporatedmaterials of U.S. Patent Pub. No. 2013/0079232. As a first example, apair of nucleotide types can be detected at the same wavelength, butdistinguished based on a difference in intensity for one member of thepair compared to the other, or based on a change to one member of thepair (e.g., via chemical modification, photochemical modification orphysical modification) that causes apparent signal to appear ordisappear compared to the signal detected for the other member of thepair. As a second example, three of four different nucleotide types canbe detected under particular conditions while a fourth nucleotide typelacks a label that is detectable under those conditions, or is minimallydetected under those conditions (e.g., minimal detection due tobackground fluorescence, etc.). Incorporation of the first threenucleotide types into a nucleic acid can be determined based on presenceof their respective signals and incorporation of the fourth nucleotidetype into the nucleic acid can be determined based on absence or minimaldetection of any signal. As a third example, one nucleotide type caninclude label(s) that are detected in two different channels, whereasother nucleotide types are detected in no more than one of the channels.The aforementioned three exemplary configurations are not consideredmutually exclusive and can be used in various combinations. An exemplaryembodiment that combines all three examples, is a fluorescent-based SBSmethod that uses a first nucleotide type that is detected in a firstchannel (e.g., dATP having a label that is detected in the first channelwhen excited by a first excitation wavelength), a second nucleotide typethat is detected in a second channel (e.g., dCTP having a label that isdetected in the second channel when excited by a second excitationwavelength), a third nucleotide type that is detected in both the firstand the second channel (e.g., dTTP having at least one label that isdetected in both channels when excited by the first and/or secondexcitation wavelength) and a fourth nucleotide type that lacks a labelthat is not, or minimally, detected in either channel (e.g., dGTP havingno label).

Further, as described in the incorporated materials of U.S. Patent Pub.No. 2013/0079232, sequencing data can be obtained using a singlechannel. In such so-called one-dye sequencing approaches, the firstnucleotide type is labeled but the label is removed after the firstimage is generated, and the second nucleotide type is labeled only aftera first image is generated. The third nucleotide type retains its labelin both the first and second images, and the fourth nucleotide typeremains unlabeled in both images.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate oligonucleotides andidentify the incorporation of such oligonucleotides. Theoligonucleotides typically have different labels that are correlatedwith the identity of a particular nucleotide in a sequence to which theoligonucleotides hybridize. As with other SBS methods, images can beobtained following treatment of an array of nucleic acid features withthe labeled sequencing reagents. Each image will show nucleic acidfeatures that have incorporated labels of a particular type. Differentfeatures will be present or absent in the different images due thedifferent sequence content of each feature, but the relative position ofthe features will remain unchanged in the images. Images obtained fromligation-based sequencing methods can be stored, processed and analyzedas set forth herein. Exemplary SBS systems and methods which can beutilized with the methods and systems described herein are described inU.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize nanopore sequencing (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”. Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). Data obtained from nanopore sequencing can be stored,processed and analyzed as set forth herein. In particular, the data canbe treated as an image in accordance with the exemplary treatment ofoptical images and other images that is set forth herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and7,211,414 (each of which is incorporated herein by reference) ornucleotide incorporations can be detected with zero-mode waveguides asdescribed, for example, in U.S. Pat. No. 7,315,019 (which isincorporated herein by reference) and using fluorescent nucleotideanalogs and engineered polymerases as described, for example, in U.S.Pat. No. 7,405,281 and U.S. Patent Pub. No. 2008/0108082 (each of whichis incorporated herein by reference). The illumination can be restrictedto a zeptoliter-scale volume around a surface-tethered polymerase suchthat incorporation of fluorescently labeled nucleotides can be observedwith low background (Levene, M. J. et al. “Zero-mode waveguides forsingle-molecule analysis at high concentrations.” Science 299, 682-686(2003); Lundquist, P. M. et al. “Parallel confocal detection of singlemolecules in real time.” Opt. Lett. 33, 1026-1028 (2008); Korlach, J. etal. “Selective aluminum passivation for targeted immobilization ofsingle DNA polymerase molecules in zero-mode waveguide nano structures.”Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures ofwhich are incorporated herein by reference in their entireties). Imagesobtained from such methods can be stored, processed and analyzed as setforth herein.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in U.S. Patent Pub. No.2009/0026082; U.S. Patent Pub. No. 2009/0127589; U.S. Patent Pub. No.2010/0137143; or U.S. Patent Pub. No. 2010/0282617, each of which isincorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readilyapplied to substrates used for detecting protons. More specifically,methods set forth herein can be used to produce clonal populations ofamplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In particular embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate. This allows convenient delivery of sequencingreagents, removal of unreacted reagents and detection of incorporationevents in a multiplex manner. In embodiments using surface-bound targetnucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to asurface in a spatially distinguishable manner. The target nucleic acidscan be bound by direct covalent attachment, attachment to a bead orother particle or binding to a polymerase or other molecule that isattached to the surface. The array can include a single copy of a targetnucleic acid at each site (also referred to as a feature) or multiplecopies having the same sequence can be present at each site or feature.Multiple copies can be produced by amplification methods such as, bridgeamplification or emulsion PCR as described in further detail below.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.

An advantage of the methods set forth herein is that they provide forrapid and efficient detection of a plurality of target nucleic acid inparallel. Accordingly the present disclosure provides integrated systemscapable of preparing and detecting nucleic acids using techniques knownin the art such as those exemplified above. Thus, an integrated systemof the present disclosure can include fluidic components capable ofdelivering amplification reagents and/or sequencing reagents to one ormore immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell canbe configured and/or used in an integrated system for detection oftarget nucleic acids. Exemplary flow cells are described, for example,in U.S. Patent Pub. No. 2010/0111768 A1 and U.S. patent application Ser.No. 13/273,666, each of which is incorporated herein by reference. Asexemplified for flow cells, one or more of the fluidic components of anintegrated system can be used for an amplification method and for adetection method. Taking a nucleic acid sequencing embodiment as anexample, one or more of the fluidic components of an integrated systemcan be used for an amplification method set forth herein and for thedelivery of sequencing reagents in a sequencing method such as thoseexemplified above. Alternatively, an integrated system can includeseparate fluidic systems to carry out amplification methods and to carryout detection methods. Examples of integrated sequencing systems thatare capable of creating amplified nucleic acids and also determining thesequence of the nucleic acids include, without limitation, the MiSeq™platform (Illumina, Inc., San Diego, Calif.) and devices described inU.S. patent application Ser. No. 13/273,666, which is incorporatedherein by reference.

4.2 Ts-Tn5 Transposomes

The invention provides transposase fusion proteins comprising a modifiedTn5 transposase and elongation factor Ts (Tsf). Ts is a protein tag thatmay be used to enhance the solubility of heterologous proteins expressedin a bacterial expression system, e.g., an E. coli expression system.Ts-Tn5 transposase has enhanced expression, solubility and purificationyields compared to an unfused standard Tn5.

The Ts-Tn5 fusion protein may be assembled into a functional transposomecomplex comprising the fusion transposase and free or appendedtransposon ends (mosaic end (ME)). In one example, Ts-Tn5 is assembledinto a functional “forktail” transposome complex. FIG. 1A illustrates aschematic diagram of an example of a forktail transposon 100. Forktailtransposon 100 comprises mosaic end (ME) sequences 110. ME sequences 110are Tn5 transposase recognition sequences used in the assembly of atransposome complex. ME sequences 110 is about 19 bp. ME sequences 110is appended with adaptor sequences 115, e.g., adaptor sequences 115 aand 115 b. Adaptor sequences 115 are relatively short oligonucleotidesequences, e.g., about 14 nucleotides. Adaptor sequences 115 are sitesfor primer annealing in, for example, a PCR amplification protocol.

Forktail transposon 100 may be assembled into a functional forktailtransposome (not shown). The forktail transposome complex may be used totagment double-stranded cDNA in an RNA-seq library preparation protocol(e.g., a TotalScript™ RNA-seq library preparation protocol). Forktailtransposon 100 obviates the need for an oligonucleotide replacement stepin the current TotalScript™ RNA-seq protocol. Forktail transposon 100may also be used in transposome complexes to eliminate the A-A and B-Bfragments that are generated in existing Nextera transposition reactions(not shown). The relatively short length of adaptor sequences 115 limitsthe renaturation of common adaptor sequences in adenaturation/renaturation process, as described in more detail withreference to FIG. 8 and FIG. 9.

In another example, Ts-Tn5 transposase is assembled into a functional“suicide” transposome complex. FIG. 1B illustrates a schematic diagram150 of assembling a Ts-Tn5 suicide transposome complex. In this example,free transposon ME sequences 110 are mixed with Ts-Tn5 transposase 160to form an assembled suicide transposome complex 165. In this example,ME sequences 110 in suicide transposome complex 165 are not appendedwith specific adaptor sequences. Because ME sequences 110 are notappended with specific adaptor sequences, DNA fragmented and taggedusing suicide transposome complex 165 are not available for subsequentamplification by specific adaptor-mediated PCR.

In one example, ME sequences 110 may be biotinylated and used toassemble a biotinylated transposome complex (not shown). Thebiotinylated transposome complex may comprise, for example, Ts-Tn5transposase or EZ-Tn5 transposase. Because ME sequences 110 arebiotinylated, double-stranded DNA fragmented and tagged using thebiotinylated transposome complex may be recovered by affinity-capturefor subsequent analysis by sequencing. The less abundant single-strandedDNA in a sample may be analyzed by sequencing the unbound fraction. Anexample of using a biotinylated transposome complex to reduce librarycomplexity is described in more detail with reference to FIG. 5.

In another example, suicide transposome complex 165 may be used in alibrary normalization process to substantially reduce the amount ofabundant nucleic acids (e.g., from rRNA) in a cDNA library, as describedin more detail with reference to FIG. 8 and FIG. 9.

To evaluate the thermal stability of transposomes assembled with astandard Tn5 transposase or Ts-Tn5 transposase, DNA was tagmented usingtransposomes that were stored at −20° C. or stored at −20° C. and thenincubated at 37° C. for 6 hours. Transposome complexes were assembledusing TotalScript transposon sequences or forktail transposon 100 ofFIG. 1. For each experiment, phage DNA (40 ng) was tagmented usingtransposomes that were stored at −20° C. or incubated at 37° C. for 6hours. Tagmented DNA was then purified using DNA Clean & Concentrator™columns (Zymo Research) and analyzed using the High Sensitivity DNAAssay (Agilent Technologies). FIG. 2A shows a plot 200 of the fragmentsize distribution of tagmented DNA prepared using transposomescomprising a standard TotalScript transposon and a standard Tn5transposase. FIG. 2B shows a plot 225 of the fragment size distributionof tagmented DNA prepared using transposomes comprising forktailtransposon 100 of FIG. 1 and a standard Tn5 transposase. FIG. 2C shows aplot 250 of the fragment size distribution of tagmented DNA preparedusing transposomes comprising the standard TotalScript transposon andTs-Tn5 transposase. FIG. 2D shows a plot 275 of the fragment sizedistribution of tagmented DNA prepared using transposomes comprisingforktail transposon 100 of FIG. 1 and Ts-Tn5 transposase. Plot 200 ofFIG. 2A, plot 225 of FIG. 2B, plot 250 of FIG. 2C, and plot 275 of FIG.2D show a line 210 of the fragment size distribution of tagmented DNAprepared using transposomes stored at −20° C. Plots 200, 225, 250, and275 also show a line 215 of the fragment size distribution of tagmentedDNA prepared using transposomes stored at −20° C. and then incubated at37° C. for 6 hours.

Referring now to FIG. 2A, line 210 of plot 200 shows that transposomes(i.e., comprising TotalScript transposon+Tn5 transposase) stored at −20°C. and then used in a tagmentation reaction generate a range of fragmentsizes. Line 215 of plot 200 shows transposomes (i.e., comprisingTotalScript transposon+Tn5 transposase) stored at −20° C. and thenincubated at 37° C. for 6 hours prior to a tagmentation reaction aresubstantially less active in fragmenting the DNA.

Referring now to FIG. 2B, line 215 of plot 225 shows that transposomes(i.e., comprising forktail transposon+Tn5 transposase) stored at −20° C.and incubated at 37° C. for 6 hours prior to a tagmentation reaction aremore active in tagmenting DNA compared to the transposomes (i.e.,comprising TotalScript transposon+Tn5 transposase) of FIG. 2A incubatedat 37° C. for 6 hours prior to tagmentation.

Referring now to FIG. 2C, plot 250 shows that transposomes (i.e.,TotalScript transposon+Ts-Tn5 transposase) stored at −20° C. (line 210)and transposomes stored at −20° C. and incubated at 37° C. for 6 hoursprior to a tagmentation reaction (line 215) have essentially the samelevel of tagmentation activity.

Referring now to FIG. 2D, plot 275 shows that transposomes (i.e.,forktail transposon+Ts-Tn5 transposase) stored at −20° C. (line 210) andtransposomes stored at −20° C. and incubated at 37° C. for 6 hours priorto a tagmentation reaction (line 215) have essentially the same level oftagmentation activity.

Referring now to FIGS. 2A through 2D, the data show that transposomescomprising Ts-Tn5 transposase (i.e., TotalScript transposon+Ts-Tn5transposase and forktail transposon+Ts-Tn5) are more stable at 37° C.(i.e., they have increased thermal stability) compared to transposomescomprising the standard Tn5 transposase.

FIG. 3A shows a plot 300 of the fragment size distribution of tagmentedDNA prepared at elevated (>55° C.) temperatures using Ts-Tn5transposomes. In this example, the Ts-Tn5 transposome comprises freetransposon ME sequences and Ts-Tn5 transposase. FIG. 3B shows a plot 350of the fragment size distribution of tagmented DNA prepared at elevated(>55° C.) temperatures using a standard Tn5 transposome (TDE1). For eachexperiment, tagmented DNA was prepared using 50 ng of human genomic DNA(Coriell), 2 pmoles of transposome, and Nextera TD buffer in a totalreaction volume of 40 μL. Reactions were incubated at 57, 65, and 70° C.for 5 minutes. Tagmented DNA was then purified using DNA Clean &Concentrator™ columns (Zymo Research) and analyzed using the HighSensitivity DNA Assay (Agilent Technologies).

Plot 300 of FIG. 3A and plot 350 of FIG. 3B show a line 310 of thefragment size distribution of DNA tagmented at 57° C., a line 315 of thefragment size distribution of DNA tagmented at 65° C., and a line 320 ofthe fragment size distribution of DNA tagmented at 70° C. Referring nowto plot 300 of FIG. 3A, the data show that at 57° C. (line 310), 65° C.(line 315), and 70° C. (line 320) the Ts-Tn5 transposome tagments theDNA generating lower molecular weight fragments. Referring now to plot350 of FIG. 3B, the data show that at 57° C. (line 310) and 65° C. (line315) the standard Tn5 transposome tagments DNA generating lowermolecular weight fragments. At 70° C. (line 320), the standard Tn5transposome has substantially reduced tagmentation activity.

FIG. 4 shows a plot 400 of DNA fragment size verses tagmentationreaction temperature for the tagmented DNA of FIG. 3A and FIG. 3B. Thedata show that the average fragment size correlates with reactiontemperature and the level of transposome activity.

Referring to FIG. 3A, FIG. 3B, and FIG. 4, the data show that theactivity of the Ts-Tn5 transposome is more thermostable compared to thestandard Tn5 transposome.

4.3 Selective Identification and Sequence Analysis of Abundant and LessAbundant Nucleic Acids

In one embodiment, the methods of the invention provide for selectiveidentification and sequence analysis of both the abundant and lessabundant (e.g., rare) species of in a DNA library. The DNA library maybe, for example, a genomic DNA library or a double-stranded cDNA libraryprepared using total RNA or mRNA. The genomic DNA library may be, forexample, a transposome-generated library or a randomly sheared and endrepaired adapter-ligated library comprising a defined adapter sequenceat its ends. Similarly, a double-stranded cDNA library may also have adefined adapter sequence at its ends, such as in a Script-Seq orExactSTRART generated library. The defined adaptor sequence maycorrespond to sequences related to primers for sequencing and/or forcluster generation in a flow cell or constitute promoter sequences forin vitro transcription.

IN one embodiment, the methods of the invention are carried out atelevated temperature. In one embodiment, the methods of the inventionuse a transposase from hemophilic organisms, for example, Thermusaquaticus, Thermococcus litoralis.

In one embodiment, the methods of the invention use a transposome (e.g.,a Ts-Tn5-ME transposome or an EZ-Tn5™ ME-Transposome (Illumina, Inc) toselectively remove abundant double-stranded DNA molecules from a sample,thereby reducing the complexity of abundant sequences in the sample. Atransposome specifically targets double-stranded DNA. Single-strandedDNA, RNA (single- or double-stranded), or RNA: DNA hybrids are notrecognized as targets by the transposome. Therefore, a transposome maybe used to selectively target double-stranded DNA molecules in a mixtureof double-stranded and single-stranded DNA molecules. For example, whena complex mixture such as a genomic DNA library or a double-strandedcDNA library is denatured by heat and subsequently cooled, highlyabundant sequences more rapidly form double-stranded structures and arerecognized as targets for transposition. The transposome may containappropriately tagged (e.g., a biotin tag) and/or appended transposonends enabling affinity-capture of the targeted double-strandedsequences. The less abundant single-stranded nucleic acids remain in thesupernatant and may be readily processed for sequence analysis. Thetagged and captured abundant DNA molecules may also be readily processedfor subsequent analysis.

FIG. 5 illustrates a flow diagram of an example of a method 500 ofreducing the abundance of double-stranded DNA in a complex library bysimultaneously fragmenting and tagging the end(s) of double-stranded DNAusing a transposome. Method 500 includes, but is not limited to, thefollowing steps.

At a step 510, a complex double-stranded DNA library is denatured. TheDNA library may be, for example, a genomic DNA library or adouble-stranded cDNA library prepared using mRNA or total RNA. In oneexample, the DNA library is denatured using the application of heat at asufficient temperature (e.g., from about 80° C. to about 95° C.) tocreate single-stranded DNA.

At a step 515, the heat-denatured DNA is renatured. For example, theheat-denatured DNA is renatured by lowering the denaturation temperature(about 80° C. to about 95° C.) to from about 40° C. to about 65° C. andincubated at about 55° C. for an extended period of time (e.g., fromabout 30 minutes to about 24 hours) sufficient to renature abundantnucleic acid species.

At a step 520, double-stranded DNA in the renatured mixture istagmented. For example, a transposome is added to the renatured mixtureand incubated at about 55° C. for up to about 10 minutes. Thetransposome may be, for example, a Ts-Tn5-ME transposome or an EZ-TN5™ME-Transposome (Illumina, Inc). The ME sequence in the transposomecomprises an affinity tag such as biotin, whereby tagmentation ofdouble-stranded DNA results in biotin-tagged double-stranded DNA. Inanother example, the biotinylated ME sequences may be appended withadaptor sequences that may be used to subsequently amplify thefragmented and tagged double-stranded DNA for sequencing.

At a step 525, the fragmented and biotin-tagged double-stranded DNA isaffinity captured to separate double-stranded DNA from single-strandedDNA. In one example, the biotin-tagged double-stranded DNA is capturedusing streptavidin-agarose. In another example, the biotin-taggeddouble-stranded DNA is captured using magnetic beads comprisingstreptavidin-agarose. Following affinity capture of the more abundantdouble-stranded DNA, the less abundant, non-biotinylated single-strandedDNA is retained in the supernatant fraction.

At a step 530, the less abundant single-stranded DNA retained in thesupernatant fraction is processed for subsequent sequencing. Forexample, the less abundant DNA may be tagged at the ends, for subsequentamplification and sequencing, either directly (e. g., Terminaltransferase) or by 3′-terminal tagging according to the methodsdescribed in the U.S. Patent Pub. No. 20050153333 and/or the U.S. PatentPub. No. 20100297643.

At an optional step 535, the captured double-stranded tagmented DNA isprocessed for amplification and sequencing. Method 500 ends.

To demonstrate the specificity of transposome-mediated fragmentation ofdouble-stranded DNA in a mixture of double-stranded DNA andsingle-stranded DNA, tagmentation reactions were performed usingsingle-stranded M13mp19 phage DNA and double-stranded pUC19 plasmid DNA.

FIG. 6 shows a photograph 600 of an agarose gel of the fragmentation ofsingle-stranded M13mp19 DNA and double-stranded pUC19 DNA by EZ-Tn5™ME-Transposome. An aliquot (20 ng) of single-stranded M13mp19 DNA ordouble-stranded pUC19 DNA in a final volume of 9 μL of buffer containing20 mM Tris.HCl (pH 8.0) and 5 mM MgCl₂ were pre-incubated at 55° C. for1 minute. After the incubation period, 1 μL of EZ-Tn5™ ME-Transposomewas added to obtain a final concentration ranging from 0.00125 μM to1.25 μM. All transposome dilutions were performed in Transposome Storagebuffer (50 mM Tris.HCl pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500mM NaCl, 0.5% NP40 and 0.5% Tween 20). Control reactions used 1 μL ofTransposome Storage buffer. The mixture was incubated at 55° C. for 5min and the reaction was stopped by adding 2 μL of stop buffer (20 mMTris.HCl pH8.0, 15% Sucrose, 66 mM EDTA, 1 SDS, 0.9% Orange G) andincubated at 70° C. for 10 minutes. An aliquot (5 μL) of each reactionwas analyzed on a 1% agarose gel and the nucleic acids were visualizedby staining with SYBR-gold. The data show that double-stranded pUC19 DNAis fragmented by the EZ-Tn5™-ME transposome; 20 ng of double-strandedpUC19 DNA is fragmented, essentially to completion using 0.125 μM EZ-Tn5ME-Transposome. The data also show that the single-stranded M13mp19 DNAis relatively intact (un-fragmented) compared to the double-strandedpUC19 DNA.

FIG. 7 shows a photograph 700 of an agarose gel of the specificfragmentation of double-stranded pUC19 DNA in a mixture comprisingsingle-stranded M13mp19 DNA and double-stranded pUC19 DNA. The reactionconditions are essentially the same as described with reference to FIG.6 except that 20 ng of single-stranded M13mp19 DNA and 20 ng ofdouble-stranded pUC19 DNA were mixed together and incubated prior to thetagmentation reaction. An aliquot (5 μL) of each reaction was analyzedon a 1% agarose gel and the nucleic acids were visualized by stainingwith SYBR-gold. The data show that reaction mixtures comprising 20 ngeach of single- and double-stranded DNA with up to 0.125 μM EZ-Tn5ME-Transposome results in specific recognition of the double-strandedpUC19 as a target and its subsequent fragmentation. The single-strandedM13mp19 DNA is left relatively intact under these conditions.

4.4 RNA-Seq Library Normalization

In one embodiment, Ts-Tn5 fusion transposases are used in thepreparation of a directional RNA-seq library for sequencing on nextgeneration sequencing platforms (e.g., Illumina GA or HiSeq platforms).For example, a first transposome complex comprising forktail transposon100 of FIG. 1A and Ts-Tn5 transposase is used to prepare tagmenteddouble-stranded cDNA. A second transposome, i.e., suicide transposome165 of FIG. 1B, comprising free ME transposon sequences (i.e., no PCRadaptor sequences) and Ts-Tn5 transposase is used to “normalize” thecDNA library (i.e., remove more abundant sequences from the library).

FIG. 8 illustrates a flow diagram of an example of a method 800 ofpreparing and normalizing an RNA library for sequencing. FIG. 9illustrates a schematic diagram 900 showing pictorially the steps ofmethod 800 of FIG. 8. Referring now to FIG. 8, method 800 includes, butis not limited to, the following steps.

At a step 810, first strand cDNA is synthesized from total RNA. Forexample, a standard TotalScript™ first strand cDNA synthesis procedureusing random hexamer primers and reverse transcriptase is used toreverse transcribe total RNA into first strand cDNA. This step is alsoshown pictorially in schematic diagram 900 of FIG. 9.

At a step 815, second strand cDNA is synthesized. For example, astandard TotalScript™ second strand synthesis procedure using UTPincorporation (instead of TTP) is used to synthesize the second cDNAstrand. This step is also shown pictorially in schematic diagram 900 ofFIG. 9.

At a step 820, double-stranded cDNA is tagmented using a forktailtransposome to generate a library of fragments. The forktail transposomecomprises forktail transposon 100 of FIG. 1A and Ts-Tn5 transposase. Thelibrary double-stranded cDNA fragments comprises abundant and lessabundant sequences. A clean-up process (e.g., AMPure XP magneticbead-based process) is performed to purify the tagmented DNA. This stepis also shown pictorially in schematic diagram 900 of FIG. 9.

At a step 825, gaps in the cDNA formed in the transposition reaction arefilled in and ligated. For example, a standard TotalScript™ gapfill/ligation reaction is used to fill-in and ligate 9 bp gaps on eachstrand of the tagmented cDNA. A clean-up process (e.g., AMPure XPmagnetic bead-based process) is performed to purify the DNA. This stepis also shown pictorially in schematic diagram 900 of FIG. 9.

At a step 830, the cDNA is PCR amplified to create short adaptorsequences. For example, PCR primers targeted to adaptor sequences 115 offorktail transposon 100 and Phusion® DNA polymerase (New EnglandBioLabs) are used to amplify the first cDNA strand. PCR primers targetedto adaptor sequences 115 are limited to the adaptor sequences and do nothybridize to the ME sequences 110 in forktail transposon 100. Theypartially overlap with the ME, but do not hyb to the entire ME Thesecond cDNA strand synthesized using UTP is not copied (amplified) bythe Phusion® polymerase. A clean-up process (e.g., AMPure XP magneticbead-based process) is performed to purify the DNA. This step is alsoshown pictorially in schematic diagram 900 of FIG. 9.

At a step 835, the cDNA library is heat denatured and renatured in alibrary normalization process. For example, the cDNA library is heatdenatured at about 95° C. for about 15 minutes and renatured at about70° C. for about 4 hours to about 8 hours. The rate at which aparticular sequence will reassociate is proportional to the number ofcopies of that sequence in the DNA sample. For example,highly-repetitive (abundant) sequence will reassociate rapidly, whilecomplex sequences (less abundant) will reassociate more slowly. The cDNAlibrary is now a mixture of single-stranded (less abundant) anddouble-stranded (abundant) DNA molecules. This step is also shownpictorially in schematic diagram 900 of FIG. 9.

At a step 840, double-stranded cDNA in the renatured reaction mixture istagmented using suicide transposome complex 165 of FIG. 1B. For example,suicide transposome 165 is added to the renatured reaction mixture andincubated at 70° C. for about 5 minutes. Renatured double-stranded DNA(e.g., from rRNA) is fragmented in the tagmentation reaction. Becausethe Ts-Tn5 suicide transposomes do not include adaptor sequences, thetagmented DNA is not amplified in subsequent process steps.Single-stranded DNA molecules (e.g., from lower abundance mRNAs) remainintact. A clean-up process (e.g., AMPure XP magnetic bead-based process)is performed to purify the DNA. This step is also shown pictorially inschematic diagram 900 of FIG. 9.

At a step 845, single-stranded cDNA is PCR amplified to create longerindexed adaptors. For example, adaptor sequences 115 are extended in thePCR reaction to comprise the remaining P5 and P7 primer sequences and anindex sequence used for sequencing. The tagmented double-stranded cDNAis not PCR amplified. This step is also shown pictorially in schematicdiagram 900 of FIG. 9.

At step 850, cDNA is PCR amplified to create the final library. Method800 ends. This step is also shown pictorially in schematic diagram 900of FIG. 9.

Throughout the process steps of method 800, the abundant sequences inthe library (e.g., from rRNA) are retained in the reaction mixture. Theabundant sequences may act as bulk carrier material to minimize sampleloss (e.g., non-specific binding to reaction surfaces) during theprocessing steps.

Method 800 provides for library preparation (step 810 through step 830)in about 6 hours, library denaturation and renaturation (step 835) inabout 4 to about 8 hours (or overnight), and library normalization (step840 through step 850) in about 3 hours. In one example, method 800 isused for preparation of a normalized RNA-seq library from about 50 pg toabout 500 pg of input RNA.

To evaluate the effect of library normalization on library output andsequencing metrics, cDNA libraries were prepared using universal humanreference RNA and method 800 of FIG. 8. Table 1 shows the sampledesignation, sample name and description of 8 RNA-seq libraries preparedwithout normalization (“NoNorm”) or with suicide-transposomenormalization (“Norm”). Control libraries (i.e., libraries that wereprepared without normalization) are designated by 582_1000 “NoNorm_A”and 582_2000 “NoNorm_B”. Libraries that were prepared usingsuicide-transposome normalization are designated by “Norm_library input(ng)_transposome input (pmol)_duplicate”. For example, the sampledesignated 582_3000 is named Norm50 ng_12 p_1 and represents a firstlibrary prepared using 50 ng of library input and 12.5 pmolsuicide-transposome (TSM). The sample designated 582_4000 is namedNorm50 ng_12 p_2 and represents a second library prepared using 50 ng oflibrary input and 12.5 pmol TSM. The sample designated 582_5000 is namedNorm100 ng_25 p_1 and represents a first library prepared using 100 ngof library input and 25 pmol TSM. The sample designated 582_6000 isnamed Norm100 ng_25 p_2 and represents a second library prepared using100 ng of library input and 25 pmol TSM. The sample designated 582_7000is named Norm50 ng_25 p_1 and represents a first library prepared using50 ng of library input and 25 pmol TSM. The sample designated 582_8000is named Norm50 ng_25 p_2 and represents a second library prepared using50 ng of library input and 25 pmol TSM. All normalization reactions wereperformed using 1× normalization buffer and a renaturation andtagmentation temperature of 68° C.

TABLE 2 RNA-seq library sample designation, name and descriptions SampleDesignation Sample Name Description 582_1000 NoNorm_A Control, NotNormalized 582_2000 NoNorm_B Control, Not Normalized 582_3000Norm50ng_12p_1 50 ng Lib, 12.5 pmol TSM, 1x Norm, buffer; 68° C.582_4000 Norm50ng_12p_2 50 ng Lib, 12.5 pmol TSM, 1x Norm, buffer; 68°C. 582_5000 Norm100ng_25p_1 100 ng Lib, 25 pmol TSM, 1x Norm. buffer;68° C. 582_6000 Norm100ng_25p_2 100 ng Lib, 25 pmol TSM, 1x Norm.buffer; 68° C. 582_7000 Norm50ng_25p_1 50 ng Lib, 25 pmol TSM, 1x Norm.buffer; 68° C. 582_8000 Norm50ng_25p_2 50 ng Lib, 25 pmol TSM, 1x Norm.buffer; 68° C.

FIG. 10 shows a bar graph 1000 of the percent sequence alignment ofcontrol and suicide-transposome normalized libraries of Table 2. Eachbar on the graph represents an RNA-seq library and shows the percent (%)aligned reads that represent normal transcripts (i.e., mRNA), % abundantreads that represent rRNA and mitochondrial RNA, and % unaligned thatrepresent material that typically does not align in an RNA-seqexperiment. The data show that all normalized samples (i.e., 582_300through 582_800) have a substantially higher percentage of aligned reads(about 85%) and a corresponding decrease in percentage of abundant readscompared to control (i.e., not normalized) libraries.

FIG. 11 shows a bar graph 1100 of the percent rRNA, mitochondrial RNAand duplicate reads in the abundant sequence fraction of samples shownin bar graph 1000 of FIG. 10. The data show that most of the abundantsequence fraction in control libraries is rRNA (i.e., about 73% of readsare rRNA). In normalized libraries, the percentage of reads from rRNA isfrom about 2 to about 6%.

FIG. 12 shows a panel 1200 of the read distributions in control andsuicide-transposome normalized libraries of Table 2. The data show thatthe coverage across transcripts in the normalized libraries is about thesame as the coverage in the control libraries.

FIG. 13 shows a panel 1300 of the alignment locations in control andsuicide-transposome normalized libraries of Table 2. The data show thatthe distribution of reads across categories (i.e., coding, UTR, Intron,and Intergenic) in the normalized libraries is about the same as thedistribution in the control libraries.

Referring now to FIGS. 10 through 13, the data show that thetransposome-mediated normalization process substantially removes rRNA,but not sequences across other RNA categories (e.g., coding, UTR,intron, and intergenic sequences).

Correlations are commonly calculated for RNA-seq data to checkexpression values and trends between samples. For example technical orbiological replicates are expected to be closely correlated, i.e., havesimilar expression values for transcripts between two samples.Correlation value falls between 0 and 1, wherein 1 is directlycorrelated, 0 is not correlated at all. Table 3 shows the data setcorrelation for the control (Ctrl_1 and Ctrl_2) and normalized librariesof FIG. 11.

TABLE 3 Data set correlation Subset Data - fpkm* in both pair has to be1 or greater, Spearman Cor, r2 Ctrl_1 1.00 Ctrl_2 0.93 1.00Norm50ng12p_1 0.93 0.92 1.00 Norm50ng12p_2 0.93 0.92 0.94 1.00Norm100ng25p_1 0.93 0.92 0.94 0.94 1.00 Norm100ng25p_2 0.93 0.92 0.940.94 0.94 1.00 Norm50ng25p_1 0.93 0.92 0.93 0.93 0.94 0.93 1.00Norm50ng25p_2 0.93 0.91 0.93 0.93 0.93 0.93 0.92 1.00 Subset Data -fpkm* in both pair has to be 1 or greater, Pearson Cor, r2 Ctrl_1 1.00Ctrl_2 0.95 1.00 Norm50ng12p_1 0.95 0.94 1.00 Norm50ng12p_2 0.95 0.940.96 1.00 Norm100ng25p_1 0.95 0.94 0.96 0.95 1.00 Norm100ng25p_2 0.950.94 0.96 0.96 0.96 1.00 Norm50ng25p_1 0.95 0.94 0.95 0.95 0.95 0.951.00 Norm50ng25p_2 0.94 0.94 0.95 0.95 0.95 0.95 0.94 1.00 *fpkm =fragment per kilobase per million reads is a commonly used method toestimate abundance value

In another application, a transposome-based library normalization methodmay be used in the analysis of nucleic acid from a single cell or asmall group of cells (e.g., about 5 to 10 cells).

In another application, a transposome-based normalization method may beused to reduce the abundance of highly repetitive sequences in a plantgenomic library.

In yet another application, a transposome-based library normalizationmethod may be used to reduce abundant sequences in a metagenomicssample. In one example, a transposome-based normalization method may beused in a “de-hosting” application to deplete abundant host sequences(e.g., human rRNA or targeted human sequences) and enrich the pathogen(e.g., viral, bacterial, etc.) content in a clinical sample (e.g.,infectious disease sample) or biological sample for sequencing.

In yet another application, a transposome-based normalization method maybe used to reduce the number of duplicates in a Methyl-seq library.

4.5 Selective Capture of Nucleic Acids from a Mixture of Other NucleicAcids or Other Impurities

In one aspect, certain nucleic acids can be selectively captured from amixture of other nucleic acids and other cellular components. In someembodiments, the nucleic acids that are selectively captured arepurified from a mixture of nucleic acids and other cellular components.In some embodiments, certain nucleic acids are selectively purified froma mixture of nucleic acids and other cellular components by removing oneor more type of other nucleic acids. In some embodiments, more than onetype of nucleic acids can be selectively captured and/or purified from amixture of nucleic acids or from a biological sample.

In some embodiments, the mixture of other nucleic acids may include ormore of double stranded DNA, single stranded DNA, double stranded RNA,single stranded RNA, RNA-DNA hybrid, partially double stranded RNA orDNA.

In some embodiments, the nucleic acids to be selectively captured and/orpurified can be double stranded DNA, single stranded DNA, RNA, doublestranded RNA. In one preferred embodiment, the nucleic acids to beselectively purified are double stranded DNA. In one embodiment, thenucleic acids to be captured and/or purified are genomic DNA. In oneembodiment, the nucleic acids to be selectively captured and/or purifiedhave highly repetitive DNA or RNA sequences. In some embodiments, thenucleic acids to be selectively captured and/or purified may havemultiple copies of the same sequence, for example, in polyploids.

In some embodiments, the nucleic acids to be selectively captured and/orpurified from a biological sample. The biological sample can be any typethat comprises the nucleic acids of interest. For example, the samplecan comprise nucleic acids in a variety of states of purification,including purified nucleic acids. However, the sample need not becompletely purified, and can comprise, for example, nucleic acids mixedwith protein, other nucleic acid species, other cellular componentsand/or any other contaminant. In some embodiments, the biological samplecomprises a mixture of nucleic acids of interest, protein, other nucleicacid species, other cellular components and/or any other contaminantpresent in approximately the same proportion as found in vivo. Forexample, in some embodiments, the components are found in the sameproportion as found in an intact cell. In some embodiments, thebiological sample has a 260/280 ratio of less than 2.0, 1.9, 1.8, 1.7,1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or less than 0.60. Insome embodiments, the biological sample has a 260/280 ratio of at least2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, orat least 0.60.

In some embodiments, specific types of nucleic acids (e.g., doublestranded DNA, single stranded DNA, single stranded RNA, double strandedRNA, RNA-DNA hybrid, etc.) can be selectively captured and/or purifiedfrom a mixture of other nucleic acids or from a biological sample byusing a nucleic acid binding protein immobilized on a solid support inwhich the protein binds selectively to the specific type of nucleicacids. In some embodiments, the nucleic acid binding protein immobilizedon a solid support have enzymatic activity and require a cofactor. Insome embodiments, the enzymatic activity may be a DNA altering activitysuch a nuclease activity, recombinase activity, ligase activity, kinaseactivity, gyrase activity, polymerase activity, transposase activity. Insome embodiments, the nucleic acid binding protein immobilized on asolid support can bind to specific types of nucleic acids in the absenceof cofactors.

In some embodiments, double stranded DNA can be selectively capturedand/or purified from a mixture of other nucleic acids and/or from abiological sample using transposome complex immobilized on solidsupport, such as beads. Transposases of the immobilized transposomecomplex specifically recognize double stranded DNA as targets. In someembodiments, double stranded DNA binding proteins such asdouble-stranded exonucleases can be immobilized on a solid support andcan be used to capture and purify double stranded DNA. The other nucleicacids such as RNA, single stranded DNA or any RND:DNA hybrids if any arenot recognized by the immobilized transposomes or the double strandedDNA binding proteins. The use of preimmobilized transposomes or otherdouble stranded DNA binding proteins on solid support allows bettercontrol by restricting the degree of freedom of transposome or otherdouble stranded DNA binding proteins.

In some embodiments, the capture and purification of double stranded DNAcan be carried out in the absence of divalent metal ions and in thepresence of EDTA. In the absence of divalent mental ions, thepreimmobilized transposomes on solid support will bind to the doublestranded DNA but will not fragment it. The captured double stranded DNAbound to transposomes immobilized on a solid support can then bepurified by removing it from the surrounding environment comprisingother nucleic acids, cellular components, proteins, organelles, etc. Insome embodiments, the captured double stranded DNA bound to thetransposomes immobilized on a solid support can be removed from thesurrounding environment by centrifugation. In some cases, the solidsupport comprise magnetic particles and the captured double stranded DNAbound to the transposomes immobilized on a solid support can be removedfrom the surrounding environment by the application of magnetic field.In some embodiments, the captured double stranded DNA bound to thetransposomes immobilized on a solid support, in which the solid supportis the wall of a tube or the flow cell, and the unbound nucleic acidsuch as single stranded DNA, RNA, RNA: DNA hybrid, excess doublestranded DNA present in the surrounding environment are removed bywashing the tube or the flow cell with wash buffer.

In some embodiments, more than one type of nucleic acid can beselectively captured and/or purified from a mixture of nucleic acids orfrom a biological sample. More than one type of nucleic acid bindingprotein immobilized on a solid support may be used to selectivelycapture more than one type of nucleic acid from a mixture of nucleicacids or from a biological sample.

In some embodiments, the methods can be further used to partially purifyselective types of nucleic acids from a mixture of nucleic acids in abiological sample. For example, a specific type of nucleic acid can beselectively removed from a mixture of nucleic acids, such as, doublestranded DNA, single stranded DNA, single stranded RNA, double strandedRNA, RNA-DNA hybrid, etc., thus unbound nucleic acids depleted of thebound nucleic acids are partially purified. In some embodiments, methodscan be used for archiving unbound nucleic acids after selectivelycapturing and removing a specific type of nucleic acid.

In some aspects, presented herein are methods of preparing animmobilized library of tagged DNA fragments comprising: (a) providing asolid support having transposome complexes immobilized thereon, whereinthe transposome complexes comprise a transposase bound to a firstpolynucleotide, the first polynucleotide comprising (i) a 3′ portioncomprising a transposon end sequence, and (ii) a first tag comprising afirst tag domain; and (b) applying a target DNA to the solid supportunder conditions whereby the target DNA is fragmented by the transposomecomplexes, and the 3′ transposon end sequence of the firstpolynucleotide is transferred to a 5′ end of at least one strand of thefragments; thereby producing an immobilized library of double-strandedfragments wherein at least one strand is 5′-tagged with the first tag.The details of such methods are disclosed in US patent applicationpublication 2014/0194324, which is incorporated by reference in itsentirety.

In some embodiments, the selective capture and/or purification of aspecific type of nucleic acid can be performed at an elevatedtemperature in which the nucleic acid binding protein immobilized on asolid support that binds selectively to the specific type of nucleicacids is stable at the elevated temperature. In some embodiments, thetemperature can be raised and rapidly cooled down allowing the moreabundant double stranded nucleic acids and the double stranded nucleicacids with highly repetitive sequences to anneal faster than otherdouble stranded nucleic acids. These annealed nucleic acids then can beselectively captured and/or removed or purified. In some embodiments,the method can be used to remove polyploidy DNA.

5 REFERENCES

-   1. Britten R J and Kohne D E (1968) Repeated sequences in DNA.    Science 161, 529-540.

6 CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of thepresent disclosure. Other embodiments having different structures andoperations do not depart from the scope of the present disclosure. Theterm “the invention” or the like is used with reference to certainspecific examples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation.

What is claimed is:
 1. A method of analyzing rare nucleic acid speciescomprising: (a) providing a library of double-stranded nucleic acids;(b) denaturing the library; (c) renaturing the library under conditionssufficient to renature abundant nucleic acid species, wherein a portionof the library comprising less abundant nucleic acid species does notrenature; (d) contacting the library with a transposase thatpreferentially binds to double-stranded nucleic acids; and (e)separating renatured abundant nucleic acid species from the lessabundant nucleic acid species.
 2. The method of claim 1, whereinseparating comprises immobilizing the transposase to a solid support. 3.The method of claim 1, wherein the transposase comprises Tn5 or TS-Tn5transposase.
 4. The method of claim 1, wherein the transposase comprisesadaptor sequences which comprise a binding moiety capable of binding toa solid support.
 5. The method of claim 4, wherein the binding moietycomprises biotin and the solid support comprises streptavidin.
 6. Themethod of claim 1, wherein the transposase comprises adaptor sequenceswhich are not compatible with downstream amplification and sequencing.7. The method of claim 1, wherein denaturing comprises heat denaturing.8. The method of claim 1, wherein renaturing comprises lowering thedenaturation temperature to a temperature of about 40° C. to about 65°C. for a sufficient period of time to renature abundant nucleic acidspecies.
 9. The method of claim 1, wherein denaturing comprises chemicaldenaturing.
 10. The method of claim 1, further comprising sequencing theseparated less abundant nucleic acid species.
 11. The method of claim 1,wherein the library comprises genomic DNA.
 12. The method of claim 1,wherein the library comprises randomly sheared DNA.
 13. The method ofclaim 1, wherein the library comprises double-stranded cDNA.
 14. Themethod of claim 1, wherein the nucleic acids in the library compriseadaptor sequences ligated to the ends of the nucleic acids.
 15. Themethod of claim 14, wherein the adaptor sequences comprise one or moreof: amplification priming binding region, sequencing primer bindingregion, and promotor sequences for in vitro transcription.
 16. Themethod of claim 1, wherein the abundant nucleic acid species comprisehighly repetitive sequences.
 17. The method of claim 1, wherein thelibrary comprises nucleic acid from a single cell or a small group ofcells.
 18. The method of claim 1, wherein the abundant nucleic acidspecies comprise host sequences and the less abundant nucleic acidspecies comprise pathogen content.
 19. The method of claim 1, whereinthe library comprises genomic RNA.